HIV-1 infection is initiated through fusion with a cell membrane, a process that is mediated by Env glycoprotein following the engagement of CD4 and coreceptors. However, there is increasing appreciation that the HIV-1 fusion process is regulated by host dependency and restriction factors, including the newly discovered SERINC5 protein that potently blocks virus entry. This realization suggests that the availability and/or abundance of host regulatory factors could determine the efficiency of HIV-1 fusion, as well as the sites of productive entry. Indeed, whereas HIV-1 fusion initiated at the cell surface does not appear to proceed to completion, this virus can mediate cell-cell fusion (?fusion-from-without?) by fusing with the plasma membranes of two adjacent cells. Our recent study has demonstrated that fusion-from-without, but not HIV-cell fusion, is highly dependent on actin dynamics, suggesting that fusion with the plasma membrane is driven by cell- generated mechanical forces. We therefore hypothesize that HIV-1 Env initiates membrane fusion but relies on a target cell to drive the energetically unfavorable enlargement of a fusion pore. This model suggests a universal mechanism for inhibition of HIV-1 fusion by SERINC5 and other restriction factors that could act through altering the properties of virus or cell membranes. The knowledge of underlying principles for promotion or inhibition of HIV-1 entry is of great importance for preventing infection and for future therapeutic strategies. Our goal is to explore the vulnerabilities of HIV-1 fusion and exploit those to develop new means to block virus entry. We propose to develop and apply novel biophysical tools to detect fusion pore dilation, delineate the determinants of productive fusion and reveal the key host factors and processes involved. Specifically, we will: (1) Develop cryo-CLEM strategies to assess the effect of HIV-1 entry sites on dilation of a fusion pore; (2) Elucidate the role of cell-generated mechanical forces in completing HIV-1 fusion at the plasma membrane and in endosomes; and (3) Delineate the mechanism by which SERINC5 inhibits HIV-1 fusion. Completion of the proposed experiments will help define the mechanisms of regulation of HIV-1 fusion (and viral fusion in general) and pave the way to developing novel strategies to prevent and treat infection.